1. Field of the Invention
The present invention relates to a method and apparatus for detecting target molecules with binding assays, such as DNA, RNA, receptor, or antibody binding assays, taking advantage of labels that produce and respond to magnetic fields.
2. Description of the Previously Published Art
Binding assays such as immunoassays, DNA hybridization assays, and receptor-based assays are widely used in the medical community as diagnostic tests for a wide range of target molecules.
As used herein, the term "analyte" indicates the molecule, species, or organism whose presence, absence, or concentration one is interested in determining, while the term "target molecule" or "target species" indicates the molecular species whose presence, absence, or concentration the assay in question actually determines. The target and analyte may be identical, or the target may be indicative of the presence or absence of the analyte. In particular, target molecules such as proteins or DNA may be a distinctive component or product of analytes such as viruses, bacteria, or other organisms, and therefore indicative of their presence.
Binding assays exploit the ability of certain molecules, herein referred to as "binding molecules", to specifically bind target molecules. Binding molecules such as antibodies, strands of polynucleic acids (DNA or RNA) and molecular receptors, are capable of selectively binding to ("recognizing") such potential target molecules as polynucleic acids, enzymes and other proteins, polymers, metal ions, and low molecular weight organic species such as toxins, illicit drugs, and explosives.
In a solid-phase binding assay, binding molecules are attached to a solid substrate, a procedure generally performed by the manufacturer of the assay. These binding molecules are referred to as "capture" molecules. When the user initiates the assay by exposing the solid substrate to a liquid sample, capture molecules immobilize target and/or label molecules on the surface via recognition events.
Through the use of labeled binding molecules, such recognition events can be made to generate a measurable signal and therefore indicate the presence or absence of a target molecule. Various types of binding assays have been devised that use radioactive, fluorescent, chemiluminescent, or enzymatic labels. Depending on the type of assay being performed, labeled binding molecules either bind to immobilized target molecules ("sandwich" assay), or compete with target molecules to bind to capture molecules ("competitive" assay). After removal of excess label, the amount of bound label is measured.
A large number of variations on the above-described binding assay methodologies have been described. For a more complete description, see a laboratory handbook such as P. Tijssen, Practice and Theory of Enzyme Immunoassays, Elsevier Science Publishers, Amsterdam, 1985, the entire contents of which are incorporated herein by reference. The common feature of all binding assays is that labeled binding molecules adhere to a solid substrate in numbers that reflect the concentration of the target molecule.
Nonspecific binding of the label, i.e. label adhering to the substrate by means other than recognition events (such as charge-charge interactions, van der Waals interactions, or adhesive contamination), is an important factor that limits the sensitivity of binding assays.
Several binding assays have been described that use magnetic particles as labels. The Force Amplified Biological Sensor (FABS) described in "Biosensor Based on Force Microscope Technology", by D. R. Baselt, G. U Lee, and R. J. Colton, J. Vac. Sci. Technol. B, vol. 14, no. 2, pp. 789-793, (1996); and in U.S. patent application Ser. No. 08/505,628, filed Jul. 21, 1995, by G. U Lee, R. J. Colton, and D. Kidwell uses a cantilever-beam force transducer to measure the total magnetic force exerted by adhering label when a magnetic field is applied.
A device described by T. Rohr in U.S. Pat. Nos. 5,445,970 and 5,445,971 uses a microbalance, rather than a cantilever-beam force transducer, to measure the force exerted by adhering magnetic label when a magnetic field is applied.
A binding assay described by R. Kotitz et al. (41.sup.st annual conference on Magnetism and Magnetic Materials, Nov. 1996; see abstract book p. 73) uses a Superconducting Quantum Interference Device (SQUID) to detect whether magnetic particles have been immobilized by biological recognition events on the side of a test tube.
The atomic force microscope (AFM) has been used by a number of research groups to measure the forces associated with recognition events. Typically, a binding molecule is attached to an AFM force transducer and a capture molecule is attached to a solid surface (or vice-versa), and the AFM is used to pull the two apart. See for example Lee et al., "Direct Measurement of the Forces Between Complimentary Strands of DNA", Science vol. 266, pp. 771-773 (1994); Hinterdorfer et al., "Detection and Localization of Individual Antibody-Antigen Recognition Events by Atomic Force Microscopy," Proc. Natl. Acad. Sci. USA vol. 93, pp. 3477-3481 (1996). In effect, such experiments detect the presence of a target molecule and discriminate specific from nonspecific interactions ("force discrimination"). Force discrimination requires that binding molecules be securely attached (i.e. covalently bonded) to the substrate and to the magnetic label. Substrate attachment is presently performed by a procedure described by G. U Lee et al., "Chemically Specific Probes for the Atomic Force Microscope," Israel J. Chem. 36, 81-87 (1996). More significantly for research purposes, such experiments could make it possible to better understand the physical basis and behavior of recognition events. In principle, it should be possible to perform the same type of study using magnetically-attracted particles rather than an AFM to pull bonds apart.
The magnetic labels generally used for FABS are beads or particles made either from nanometer-sized iron oxide crystallites, polymer impregnated with nanometer-sized iron oxide crystallites, or porous glass filled with iron oxide crystallites. Such particles are commonly used for magnetic separation in molecular biology and are manufactured by several firms, including Dynal, Inc., Lake Success, N.Y.; Bangs Laboratories, Inc., Carmel, IN; CPG, Inc., Lincoln Park, N.J.; and PerSeptive Biosystems, Framingham, Mass. These particles can be obtained with surface functional groups that may be used to immobilize molecules such as streptavidin, antibodies, or DNA. The particles are paramagnetic; that is, their magnetization is a function of the external magnetic field, and when the field is removed, the magnetization of the particles settles to zero. This "relaxation" does not happen instantly, but occurs over a period typically measured in microseconds or milliseconds, depending on the size of the iron oxide crystallites. Particles based on nanometer-sized iron oxide crystallites are sometimes termed "superparamagnetic", since their magnetization in a given magnetic field tends to be much greater than normal paramagnetic materials.
Particles fabricated from ferromagnetic materials (such as NdFeB or nickel) or ferrimagnetic materials (such as micron-sized iron oxide or ferrite particles) can also be used as magnetic labels. Both types of materials can be magnetized to a substantially greater magnetic moment than superparamagnetic particles, but they also retain their magnetism in the absence of an external magnetic field. Since magnetized particles aggregate and thus cannot be used in a FABS assay, the particles must be obtained in a nonmagnetic state and kept nonmagnetic until they are immobilized by the antibody-antigen interactions. These materials therefore present certain development challenges.
3. Objects of the Invention
It is an object of this invention to selectively detect a wide range of chemical and biological species obtained from either the vapor or liquid phase, with a high degree of sensitivity.
It is a further object of this invention to simultaneously detect numerous chemical or biological species in a single assay.
It is a further object of this invention to rapidly detect chemical and biological species, on the order of 15-30 minutes per assay.
It is a further object of this invention to produce a sensor for chemical and biological species in the form of a compact, fully automated device.
It is a further object of this invention to measure intermolecular binding forces and thereby analyze recognition events.
These and further objects of the invention will become apparent as the description of the invention proceeds.